The Role Of JNK Signalling In Alveolar Type â…¡ Cells Apoptosis Under Oxidative Stress

Part one Isolation, purification, culture and identification of primary rat alveolar type II epithelial cells in vitro BackgroundThe alveolar epithelium is vulnerable to various kinds of stress, and its repair following injury mainly depends on the ability of its stem cells, the alveolar typeΙΙepithelial cells (ATII cells). ATII cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium and could restore the integrity of the alveolar epithelial by proliferation, spreading, and migration, and finally by differentiation to alveolar type I cells, restoring the normal morphology and functional properties of the alveolar epithelium. Moreover, ATII cells have some crucial functions, which include: synthesis, secretion, and recycling of surfactant; transport of ions and water; synthesis of immune effector molecules. But the technology of isolating, and culturing ATII cells is complicated. The aim of this study is to obtain the method of isolating, purifying and culturing ATII cells from adult rat and to provide enough and highly purified ATII cells for next research. ObjectiveTo establish the primary culture technology and identification method for alveolar type II epithelial cells. Providing experiment means for obtaining highly purified ATII cells to investigate the molecular mechanisms of oxidative stress-induced alveolar epithelial cell apoptosis. MethodsATII cells were isolated from anesthetized (Chloral Hydrate) male Sprague–Dawley rats (≈200 g). Firstly, lungs were cleared of blood by saline perfusion and removed from the thorax. After lavage, the lungs were instilled with 0.08% trypsin solution, incubated for 20 min at 37°C in an incubator containing 5% CO2. Dissected the lung parenchyma away from the major airways and minced the lung pieces with a scissors to a final size of ~1 mm3 in the presence of DNase I (250μg/ml) and fetal bovine serum (FBS). The cell suspension was sequentially filtered through copper meshes and centrifuged at 1500 rpm for 10 min. The cell pellet was resuspended in DMEM/F12 and panned on an IgG-coated plastic dish at 37°C to remove macrophages. The unattached cells were centrifuged and suspended in DMEM/F12 containing 10% FCS, 100 units/ml penicillin, 100μg/ml streptomycin, seeded on 6-well tissue culture plates and cultured in 95% humidified air and 5% CO2 at 37°C. Cells were used about 36 h thereafter. The cell viability was assessed by the method of tryban blue staining. The cells were identified under TEM. The purity of the culture cells was assayed with papanicolaou Staining and the morphological changes of ATII cells after culturing various times (36, 72, 96 and 120 h) were observed in vitro.Results1. About 2.0×107 cells were obtained from one adult rat.2. The lamellar body of ATII cells was observed under transmitting electron microscopy (TEM) and the purity of the culture cell was > 90% assayed with modified papanicolaou Staining.3. The cell viability was > 90% assessed by the method of tryban blue staining.4. Cells grown well and there were many granules in cytoplasm after 36 h to 72 h of culture. Cells became rhomboid, the number of granules was decreased and vacuoles presented in cytoplasm after more than 96 h of culture.Conclusions1. Trypsin digestion and specific immunosorption to plates coated with IgG were the efficient methods to isolate, and purify ATII cells. The cells exhibiting high yield, purity and viability can meet the experiment need.2. ATII cells exhibit excellent growth property after 36h to 72 h of culture and this term is good for in vitro study. Part two Injury of alveolar type II cells and JNK signal transduction induced by hydrogen peroxideBackgroundOxygen therapy is an effective and often used method for treating acute respiratory failure. Unfortunately, exposure to hyperoxia with long term may result in oxidative lung injury. The production of reactive oxygen species (ROS) during exposure to hyperoxia is widely thought to be responsible for oxidative lung injury. ROS could damage the cell, including membrane lipid peroxidation, oxidative modification of proteins, and the nicking of the DNA, resulting in the impairment of cellular integrity and function. Furthermore, it is known that intracellular signaling cascades which lead to cell apoptosis can be activated by ROS. The mitogen-activated protein kinases (MAPKs) including ERK, p38 MAPK and JNK are the family of kinases that transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli. The JNK pathway is a major stress signaling pathway in cells that plays important role in many cellular processes, including development, apoptosis, growth and immune responses, but the role of JNK is cell type and stimulus dependent. The effect of JNK signaling probably involve in oxidative stress-mediated ATΙΙcells apoptosis, which may occur during lung injury and fibrosis, has not been clearly defined.Objective1. To establish an oxidative stress model of primary ATII cells in vitro with H2O2 and culture medium.2. To evaluate the model by investigating the change of cellular shapes, viability, apoptosis and ultrastructure induce by H2O2 and to explore the dose- and time- dependent effects of H2O2 on the viability and apoptosis of ATII cells.3. To study the JNK activation during H2O2 stimulating and the role of JNK signaling in ATII cells apoptosis.MethodsATⅡcells were isolated, purified and primary cultured from the healthy cleaning type Spraque-Dawley adult rats, the cell viability and purity was determinated through trypan blue staining and modified papanicolaou's staining, respectively. To study the dose- and time- dependent effects of H2O2, different dose of H2O2 (0, 50, 100, 500 and 1000μM) treated primary rat ATΙΙcells for 3 h and 500μM H2O2 was added into cells for various times (0, 1, 3, 6 and 9 h). The changes of cellular morphology, cell viability and apoptosis were detected by inverted phase contrast microscope, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. The ultrastructure changes were observed under and transmission electron microscopy (TEM) after ATΙΙcells treated with 500μM H2O2 for 3 h. The expression of p-JNK was detected by western blot after ATΙΙcells treated with 500μM H2O2 for various times (0, 5, 10, 30, 180, 360 and 540 min). To study the role of JNK signaling in ATII cells apoptosis, ATΙΙcells were randomly divided into four groups: control;CON plus SP600125 (25μM); H2O2 (500μM);H2O2 (500μM) plus SP600125 pretreatment. The cell viability and apoptosis were measured in each group. The nuclei changes induced by H2O2 were observed under fluorescence microscope after DAPI staining.Results1. Compared with control group (0μM H2O2), 50μM H2O2-treated group showed no morphological changes. Intercellular space slightly widened when cells were treated with 100μM H2O2. With the increase of H2O2 concentration, intercellular space became wider, cells turned smaller, nucleus became condensable and the number of cytoplasmic granules was decreased. Cells turned round, shrinked, ruptured when treated with 1000μM H2O2. The degree of cell damage was also increased with the time of 500μM H2O2 incubation prolonged.2. The morphology of apoptotic cell was detected in the cells treated with 500μM H2O2 for 3 h by TEM.3. Compared with the control group (0μM H2O2), the dose of 50μM and 100μM of H2O2 presented no obvious cytotoxity to ATII cells(P > 0.05), but the dose of 500μM and 1000μM of H2O2 significantly decreased the cell viability(P < 0.05).4. Compared with the control group (0 h), the cell apoptosis was gradually increased and the cell viability was gradually decreased with the time of H2O2 incubation prolonged.5. JNK was rapidly activated by 500μM H2O2 treatment, p-JNK peaked at 30 min and decreased gradually. Pretreatment ATII cells with SP600125 could effectively inhibited JNK activation-induced by H2O2 treatment.6. SP600125 pretreatment effectively enhanced cell viability and decreased apoptotic rate under oxidative stress.7. Normal cell nuclei appeared large, around and emiting light blue fluorescence, while nuclei became small and condensed and often exhibit a halo effect after H2O2 treatment observed under fluorescence microscope with DAPI staining. SP600125 pretreatment effectively protected cell nuclei under oxidative stress.Conclusions1. H2O2 treatment can induce morphological changes in ATⅡcells.2. H2O2 induced ATⅡcells damage in a dose- and time- dependent manner.3. ATⅡcells apoptosis induced by H2O2 was confirmed by TEM and flow cytometry.4. JNK may play a proapoptotic role in the apoptosis of ATⅡcells induced by H2O2. Part three JNK modulate the expression of apoptosis related proteins in ATII cells under oxidative stressBackgroudThe production of ROS during exposure to hyperoxia is widely thought to be responsible for oxidative lung injury. Excessive ROS could result in cell apoptosis in lung tissue, and the degree of lung injury is related to the apoptosis of epithelial and endothelial cells. Recently, the modulation of ROS in cell apoptosis was widely studied. ATⅡcells are very important to maintain the normal respiratory function and to restore the integrality of alveolar epithelium. If ATII cells were intensively damaged, lung injury or tissue fibrosis would inevitably occur. There are more and more researchers paying their attention to the apoptosis of ATII cells, but the molecular mechanisms of ROS-induced alveolar epithelial cell death remain incompletely understood. The aim of this study is to explore the effect of ROS on apoptosis related protein expression and nuclear factor activation in ATII cells. Moreover, using SP600125 to study the possible role of JNK in regulating the expression of apoptosis related protein and the activation of nuclear factor under oxidative stress. Objective 1. To observe the destructive effect of H2O2 on mitochondrial membrane potential (MMP).2. To explore the effect of H2O2 on the expression of apoptosis related protein and the activation of nuclear factor in ATII cells.3. To study the possible roles of JNK in regulating the expression of apoptosis related proteins and the activation of nuclear factor under oxidative stress.MethodsThe MMP changes induced by 500μM H2O2 treatment were observed under fluorescence microscope and measured by flow cytometry after JC-1 staining. The expression of apoptosis related proteins (Bax, p53, active caspase-3) and the activation of NF-κB induced by 500μM H2O2 treatment were detected by western blot. The mitochondrias were stained by Mitotracker Red; the expression and location of p53 and NF-κBp 65 was detected by immunofluorescence and observed under confocal microscopy.Results1. There was only red JC-1 fluorescence in normal ATII cells. After H2O2 treatment, ATII cells began to give off green JC-1 fluorescence and the intensity of red JC-1 fluorescence became weaking. Fluorescence ratio between red and green was decreasing with the increase of H2O2 treatment time detected by flow cytometry after JC-1 staining.2. Compared with control group, the expression of Bax,p53 and activated caspase-3 were increased after 500μM H2O2 stimulating measured by western blot and their expression were obviously enhanced with the treatment time prolong. SP600125 pretreatment effectively inhibited the expression of Bax,p53 and activated caspase-3 and the activation of NF-κB.3. Using confocal microscopy, we corroborated that H2O2 treatment increased the expression of p53 and NF-κB p65 and nuclei translocation of both is evident in the merged images. Pretreatment ATII cells with SP600125 effectively inhibited their expression and nuclei translocation induced by H2O2 treatment.Conclusions1. H2O2 treatment impaired MMP in ATII cells, and the impairments were aggravated with the treatment time prolonged.2. H2O2 treatment upregulated the expression of apoptosis related proteins (Bax,p53 and activated caspase-3) and and the activation of NF-κB through JNK signaling.Part four The protective effects of N-acetylcysteine on oxidative stress-induced alveolar typeΙΙepithelial cell injury BackgroundSince ROS play an important role in cell death under oxidative stress, we strongly speculate that antioxidant therapy would seem to be helpful to preventing oxidative stress-induced ATΙΙcells apoptosis. NAC acts as a precursor of GSH synthesis, has frequently been shown to be an effective antioxidant in some type of cells under oxidative stress. But it is still unknown whether NAC treatment can reduce oxidative stress-induced apoptosis in ATΙΙcells, so in this study we investigate effects of NAC on MAPKs activation and cell damage-induced by H2O2 treatment in AT cells.Objective1. To examine whether the administration of antioxidant N-acetylcysteine (NAC) would provide protection against oxidative stress-induced damage to ATII cells.2. To explore the effect of NAC on MAPKs activation and intracellular ROS generation-induced by H2O2 treatment.Methodsprimary rat ATΙΙcells cultured in vitro were randomly divided into four groups: control, NAC (5mmol/L), H2O2 (500μmol/L), H2O2 plus NAC pretreatment groups. Cell viability were assessed with MTT assay. Apoptotic ratio and intracellular ROS levels were measured by flow cytometry. The phosphorylation of MAPK were analysed by western blot. Results 1. Compared with control group, decreased cell viability, increased apoptotic rate were observed after the stimulation of H2O2 for 3 h (Both P < 0.05).2. Compared with control group, intracellular ROS level was increased (P < 0.05) in H2O2 group. The expression of p-JNK, p-ERK and p-p38 was increased after H2O2 treatment.3. Pretreatment with NAC could effectively improved cell viability, decreased apoptosis under oxidative stress (all P < 0.05).4. NAC could attenuate the phosphorylation of JNK and the generation of intracellular ROS-induced by H2O2 treatment (P < 0.05).ConclusionsNAC effectively attenuate cell apoptosis and improve cell viability through eliminating intracellular ROS and inhibiting MAPKs activation.